CH628330A5 - METHOD FOR REMOVING ONE OR MORE PROTECTIVE GROUP (S) FROM AMINO ACIDS OR PEPTIDES WITH ONE OR MORE THIOL GROUP (N). - Google Patents

Description

The present invention relates to a method. The peptides which can be used for the present invention can be

for the removal of one or more protective group (s) also contain residues other than amino acid residues, provided that an amino acid or a peptide with one or more 35 peptides contains one or more protected thiol group (s).

Thiol group (s), which have p-methoxybenzyl, 1-adamantyl or.

tert-butyl is or are protected. As examples of mercury salts of carboxylic acids of the form

In general, it is common for organic reactions to be the mercuric salts of acetic acid, dichloroacetic acid.

required to protect a thiol group and then to regenerate acid, trichloroacetic acid and trifluoroacetic acid.

rieren. Protection and 4 <> are mentioned in particular in peptide synthesis, the mercuric salts of acetic acid and trifluoro-

the regeneration of thiol groups is quite important. acetic acid are particularly preferred. Such a Mercuri salt

The most commonly used methods for protecting egg, based on the amino acid or peptide, can be in a thiol group and then removing the amount of e.g. 1 to about 4 molar equivalents are used

Protection group are nowadays the protection of the thiol group.

a benzyl group and the subsequent removal of this "5 in the practice of the invention can take place

Protective group with metallic sodium in liquid ammonium or the direct addition of such a mercuric salt to the reac-

niak and the protection of the thiol group with p-methoxybenzyl and ion mixture, the materials are added which are able to form the subsequent removal of the latter group with the mercuric salt in the reaction mixture. E.g.

Refluxing trifluoroacetic acid or refluxing it can mercuric oxide dissolved in a carboxylic acid of formula I the anhydrous hydrogen fluoride. [J. Biol. Chem., Vol. 50, so that the resulting mercuri salt is used for the purposes

108, 753-761 (1935), Bull. Chem. Soc. Japan, Vol. 37, No. 3, the invention can be used.

433-4-34 (1964) and Bull Chem. Soc. Japan, Vol. 40,2164- The method according to the invention can normally be found in

2167 (1967)]. But because they are designed to remove this protective presence of a solvent. The Lö-

conditions applied to groups are quite drastic, e.g. be a carboxylic acid of formula I, e.g.

cases in which it turns out to be impossible to use thiol protection- 55 acetic acid, dichloroacetic acid, trichloroacetic acid or trifluoro-

to selectively remove the group while the other functional acetic acid, or a mixture of such acid with water,

groups and protective groups remain intact. It is also possible to use other solvents, such as water,

Because tert-butyl and 1-adamantyl groups are easily incorporated into the alcohols, e.g. Methanol, ethanol, propanol or butanol,

Thiol group can be introduced, attempts have been un- and mixtures of such alcohols with water, further Te-

taken as protective groups for thiol groups, such as trahydrofuran, dioxane, dimethylformamide, chloroform, aceto

turn, however, it was only found that these tonitrile, formic acid and mixtures thereof with water

It is very difficult to remove protective groups afterwards [J. A. C. S. to choose a solvent that combines the desired compound

85: 201-207 (1963)]. This means that no effective comparison has so far been easy to solve and that

drive to remove these protecting groups. suitable reaction. The method according to the invention can

Under these circumstances, a new process for 65 at an appropriate temperature, e.g. in the range of approx.

Regeneration of a by p-methoxybenzyl, 1-adamantyl or -20 to +100 ° C, preferably from about 0 to about 80 ° C,

Tert-butyl protected thiol group searched, whereby leads are found.

was that these protective groups with extreme ease and The treatment time in the inventive method

3rd

628 330

depends on various factors such as the type and amount of the amino acid or peptide used, the solvent used and the mercuri salt used, but in many cases it is sufficient to carry out the treatment for about 30 seconds to 12 hours. 5

The thiol protecting group, i.e. According to the invention, p-methoxybenzyl, 1-adamantyl or tert-butyl can be removed in this way, but in certain cases the amino acid or the peptide can be in the form of a mercaptide, the free thiol group using a process known per se. io rens can be regenerated. E.g. the mercaptide is dissolved in water, an aqueous solvent or a pure solvent, e.g. Formic acid, acetic acid, dimethylformamide or tetrahydrofuran, dissolved or suspended and mixed with a mercapto compound, e.g. Hydrogen sulfide, mercaptoethane 15 noi, thioglycolic acid, sodium sulfide or ammonium sulfide, treated, whereby the free thiol group is regenerated.

The method according to the invention has the following advantages:

1. The removal of the protective groups is achieved under extremely mild conditions.

2. The protective groups can be removed in good yield.

3. The procedure and post-treatment are easy and simple. 25th

4. The protecting group on the thiol group can be removed selectively, leaving other functional and protecting groups intact.

In the following, examples for the production of amino acids or peptides with a protected thiol group or 30 protected thiol groups are given as reference examples and examples for the removal of the protective groups as exemplary embodiments.

When abbreviations are used in this specification to denote amino acids, peptides, protective groups, etc., the nomenclature prescribed by IUP AC IUB and the common names commonly used in this particular field are used. If any amino acid can be in the D and L configurations, any reference to that acid will refer to the L form unless the D form is specifically mentioned.

tBu

I.

H-Cys-OH

Using L-cysteine and tert-butanol, the procedure of Reference Example 1 is repeated to produce the above compound.

Melting point: 244.0 to 245.0 ° C; [ajg '-15.4 ° (c = 0.57, glacial acetic acid);

Elemental analysis for C7H1502NS

Calculated: C45.13; H8.65; N7.52; S 17.22;

Found: C 45.11; H8.32; N7.47; S 17.77.

Reference example 3

Preparation of the dicyclohexylamine salt of tert-butoxy-carbonyl-S-tert-butyl-L-cysteine of the formula:

t-Bu

I.

Boc-Cys-OH • DCHA

In a conventional manner, the S-tert-butyl-L-cysteine of the formula:

tBu

I.

H-Cys-OH

mittert.-Butyl-S-4,6-dimethylpyrimidin-2-ylthiol carbonate tert-butoxycarbonylated and then crystallized as the dicyclohexylamine salt.

Yield: 90%; Melting point: 181.0 to 182.5 ° C;

[a] β + 7.4 ° (c = 0.83, methanol);

Elemental analysis for C24H4604N2S

Calculated: C 62.93; H 10.10; N6.10; S 6.99;

Found: C 62.92; H 10.36; N5.95; S 7.05.

The dicyclohexylamine salt of tert-butoxycarbonyl-S-1-adamantyl-L-cysteine of the formula:

Reference example 1

Preparation of S-l-Adamantyl-L-cysteine of the formula:

Adm

I.

H-Cys-OH

12.1 g of L-cysteine are dissolved in 120 ml of trifluoroacetic acid, whereupon 15.2 g of 1-adamantanol are added. The mixture is stirred at room temperature for 12 hours. The trifluoroacetic acid is distilled off under reduced pressure. The oily residue is dissolved in 200 ml of water and cooled with ice. The solution is then brought to pH = 6.0 with concentrated aqueous ammonia, whereupon crystals separate out. The crystals are isolated by filtration, rinsed with water, ethanol and ether and recrystallized from water.

Yield: 23.0 g; Melting point: 227.0 to 228.0 ° C (decomposition); [cx] d - 14.5 ° C (c = 0.51, glacial acetic acid);

Elemental Analysis for C13H2202NS Calculated: C60.90; H8.65; N5.46; S 12.51; Found: C 59.51; H 8.16; N5.41; S 12.33.

Reference example 2

Preparation of S-tert-butyl-L-cysteine of the formula:

Adm

I.

45 Boc-Cys-OH DCHA

is made in the same manner as above.

Yield: 71.4%; Melting point: 173.0 to 174.0 ° C; 50 Md + 5.8 ° (c = 0.55, methanol);

Elemental analysis for C30H53O4N2S Calculated: C 67.00; H 9.93; N5.21; S 5.96; Found: C67.09; H9.88; N5.08; S 5.85.

55 The tert-butoxycarbonyl-S-p-methoxybenzyl-L-cysteine used has the formula:

MOB

I.

Boc-Cys-OH

60

65

is a commercially available product.

Reference example 4

Preparation of the compound of the formula:

tBu

I.

Boc-Gly-Cys-Gly-OMe

628 330

4th

(1) Preparation of the compound of the formula:

tBu

I.

Boc-Cys-Gly-OMe

3.0 g of glycine methyl ester hydrochloride are dissolved in 30 ml of chloroform, and 3.36 ml of triethylamine are added with ice cooling. This mixture is mixed with the N-hydroxy-5-norbornene-2,3-dicarboximide ester (HONB ester) of the compound of the formula:

tBu I

Boc-Cys-OH

(prepared from 9.2 g of the compound of the formula:

tBu

I.

Boc-Cys-OH DCHA

and 3.6 g of HONB in dioxane with 4.1 g of dicyclohexylcarbodiimide as the condensing agent), and the mixture is stirred for 12 hours at room temperature. The chloroform is distilled off under reduced pressure and the oily residue is dissolved in 24 to 150 ml of acetic acid and washed with a 4% aqueous sodium hydrogen carbonate solution, a saturated aqueous citric acid solution and finally water. The ethyl acetate is then evaporated and the residue is crystallized by adding water.

Yield: 5.9 g; Melting point 73.0 to 75.0 ° C; [a] ^ - 17.9 ° (c = 0.56, dimethylformamide);

Elemental Analysis for Q5H28O5N2S Calculated: C 51.70; H 8.10; N8.04; S 9.20; Found: C51.70; H 8.12; N8.05; S 9.14.

(2) Preparation of the compound of the formula:

tBu I

Z-Gly-Cy-Gly-OMe

5.23 g of the compound of the formula:

tBu

I.

Boc-C ys-Gly-OMe are dissolved in 30 ml of trifluoroacetic acid and the solution is shaken for 15 minutes at room temperature. The trifluoroacetic acid is evaporated off under reduced pressure and the residue is dried well. It is then dissolved in 30 ml of dimethylformamide and mixed with 7.0 ml of triethylamine with ice cooling. After adding 5.8 g of the compound of the formula Z-Gly-ONB, the mixture is stirred for 48 hours. The dimethylformamide is evaporated under reduced pressure and the residue is dissolved in ethyl acetate. The solution is washed with a 4% aqueous sodium hydrogen carbonate solution, 1 normal hydrochloric acid and water and then evaporated to dryness under reduced pressure. Petroleum ether is added to the residue, and the mixture is then left to stand, after which crystals separate out. The crystals are isolated by filtration.

Yield 6.1 g; Melting point: cannot be determined because the substance is liquid at room temperature; [a] f? - 11.4 ° (c = 0.46, dimethylformamide);

Elemental analysis for C2oH2906N3S

Calculated: C 54.65; H 6.65; N9.56; S 7.30;

Found: C55, ll; H6.60; N9.48; S 6.55.

5 The compound of the formula:

MOB

I.

Z-Gly-Cys-Gly-OMe

10th

is also made in the same manner as above. Melting point: 90.0 to 95.0 ° C; [a] o - 23.1 ° (c = 0.55, dimethylformamide);

Elemental analysis for C24H2y07N3S

15 Calculated: C 57.20; H 5.80; N8.34; S 6.37;

Found: C 57.60; H 5.81; N8.23; S 6.36.

example 1

20 Recovery of cysteine from S-protected cysteine

1) 1 millimole of S-protected cysteine is dissolved in 10 ml of trifluoroacetic acid, after which 0.2 ml of anisole is added. The mixture is cooled to 0 ° C. and 319 mg (1 millimole) of Hg (CH3 COO) 2 are added. The mixture is stirred at 0 ° C for 15 minutes. The trifluoroacetic acid is evaporated under reduced pressure over 5 minutes, after which 40 ml of water are added to the residue. The mixture is washed once with ether. Hydrogen sulfide gas is bubbled into this solution for 30 minutes and the resulting mercury is filtered off, after which the filtrate is concentrated. The residue obtained is dissolved in a small amount of water and the water is evaporated to remove the excess acid. This procedure is repeated three times and the residue obtained at the end is analyzed with the aid of an amino-35 acid analyzer. The results are shown in the following table. The S-benzyl group is quite stable under the above conditions.

tBu

Adm

MOB

40

50

55

60

65

S-protected H-Cys-OH H-Cys-OH H-Cys-OH

amino acid

% Recovery 99.8% 102.0% 100.8%

of cysteine

No deprotection takes place when the above reaction is carried out using the acetate of Ag, Cu, Zn, Ni or Pb instead of Hg (CH3COO) 2.

2) 1 millimole of S-protected cysteine is dissolved in 10 ml of 80% formic acid or 80% acetic acid, after which 0.2 ml of anisole is added. At room temperature, 319 mg (1 millimole) of Hg (CH3COO) 2 are added and the mixture is stirred for 1 hour. The reaction mixture is treated and analyzed in the same way as under 1).

The results are shown in the following table:

MOB

tBu

S-protected amino acid solvent

80% formic acid 85.0% 80% acetic acid 73.0%

Adm

I I I

H-Cys-OH H-Cys-OH H-Cys-HO

86.0% 48.0%

80.0%

3) 1 millimole of S-protected cysteine is dissolved in 20 ml of 80% acetic acid, then 0.2 ml of anisole and then 512 mg (1.2 millimoles) of Hg (CF3COO) 2 are added. The mixture is stirred at room temperature for 1 hour.

5

628 330

The reaction mixture is then treated and analyzed in the same manner as under 1).

The results are shown in the following table:

MOB tBu

I I

S-protected H-Cys-OH H-Cys-OH

Amino acid% recovery of cysteine 94.0% 92.0%

If a solution of Hg (CCl3COO) 2 is used instead of Hg (CF3COO) 2, the percentage recovery of cysteine from the compound of the formula is:

MOB

I.

H-Cys-OH 96.0%.

Example 2

Preparation of the compound of the formula Z-Gly-Cys ~ Gly-OMe 1) From the compound of the formula:

5 MOB

I.

Z-Gly-Cys-Gly-OMe

3.02 g of the compound of the formula:

10th

MOB

I.

Z-Gly-Cys-Gly-OMe

15 are dissolved in 45 ml of cold trifluoroacetic acid, after which 1.5 ml of anisole are added. After adding 1.91 gHg (CH3COO) 2 at 0 ° C, the mixture is stirred for 15 minutes. The trifluoroacetic acid is distilled off under reduced pressure and ether is added to the residue. The resulting precipitate is isolated by filtration.

Yield: 4.1 g;

Elemental analysis for Ci8H2oOgN3SF3Hg;

Calculated: C 31.06; H 2.90; N6.04; S 4.61; Hg 28.32; F 8.19.

Found: C31.02; H2.87; N6.18; S4.60; Hg28.68; F8.18.

2.0 g of the above mercaptide are dissolved in 30 ml of dimethylfor-30 of the compound of the formula Z — Gly-Cys-Gly-OMe, the mamide, followed by 80 minutes of sulphurous water by treating the compound of the formula:

lets substance gas bubble into the solution. The resulting HgS is filtered off with Celite as a filter aid and the filtrate under MOB

reduced pressure to dryness. The crystalline I

Water is added to the residue, whereupon the crystals are isolated by 35 Z-Gly-Cys-Gly-OMe filtration and recrystallized from methanol and water. can be obtained with Hg (CF3COO) 2 in 80% acetic acid.

Yield: 1.1g (100%); Melting point 130 to 132 ° C; 1 millimole of the compound of the formula:

D - C * 7 ° (n - D II Ìi m afïntïînmi \ 40

[Ujlf, Jj / VjJ / j JL / lHlWUlJ> UVllliaiIIlU ^

Elemental analysis for C16H2i06N3:

Calculated: C 50.12; H 5.52; N 10.96; S 8.36.

Found: C49.61; H 5.18; N 10.97; S 8.44.

2) From the compound of the formula:

tBu

I.

Z-Gly-C ys-Gly-OMe

By treating the compound of the formula:

tBu

I.

Z-Gly-C ys-Gly-OMe in the same manner as in 1) gives the mercaptide and Z-Gly-Cys-Gly-OMe in yields of 84.2% and 84.5%, respectively.

This compound is identical in melting point and the value of [a] D to the compound obtained under 1).

Elemental analysis:

Calculated: C50.12; H5.52; N10.96; S8.36. Found: C50.13; H5.61; N 10.77; S8.27.

3) The following table gives the yields of crystals

MOB

I.

Z-Gly-Cys-Gly-OMe

45 is dissolved in 20 ml of 80% acetic acid, after which 512 mg (1.2 millimoles) or 853 mg (2.0 millimoles) Hg (CF3COO) 2 are added. The mixture is stirred at room temperature for 60 minutes. After adding 40 ml of 80% acetic acid, hydrogen sulfide gas is bubbled into the mixture for 20 minutes. The HgS is filtered off and the filtrate is evaporated to dryness under reduced pressure. Water is added to the residue, whereupon the resulting crystals are isolated by filtration and recrystallized from ethyl acetate.

55 Hg (CF3COO) 2 1.2 molar equiv. 2.0 molar equiv. Yield 83.9% 76.5%

When the above reaction process is carried out using 1.2 molar equivalents of Hg (CCl3COO) 2 instead of 60 Hg (CF3COO) 2, the yield of the compound of the formula Z-Gly-Cys-Gly-OMe is 74.9%.

65

Example 3

Preparation of bis-tert-butoxycarbonyl-L-cystine 3.41 g of the compound of the formula:

628 330

6

MOB

!

BOC-Cys-OH

are dissolved in 200 ml of 80% aqueous methanol, and after adding 5.12 g of Hg (CF3COO) 2, the solution is stirred at room temperature for 2 hours. After adding 100 ml of 80% aqueous methanol, hydrogen sulfide gas is bubbled into the reaction mixture for 30 minutes. The resulting HgS is filtered off and the filtrate is concentrated to half its initial volume under reduced pressure. The residue is mixed with 4.2 g of sodium hydrogen carbonate and the mixture is further concentrated under reduced pressure. After the methanol was removed, 100 ml of water was added to the residue, followed by oxidation by bubbling air over 40 hours. The mixture is acidified with citric acid and the resulting oil extracted twice with 150 ml of ethyl acetate. The ethyl acetate is distilled off and the residue is crystallized by adding petroleum ether and isolated by filtration. The crystalline product is recrystallized from ethyl acetate.

Yield: 1.85 g; Melting point: 149.0 to 150.0 ° C (decomposition); [a] ^ - 136.4 ° (c = 1.84 in methanol); Elemental analysis for C16H28OgN2S2:

Calculated: C 43.62; H 6.48; N6.36; S 14.55. Found: C43.72; H6.34; N6.24; S 14.25.

Example 4

Production of oxytocin

1) Preparation of the compound of the formula Boc-Cts (MOB) -Tyr-Ile-Gln-Asn-Cys (MOB) -Pro-Leu-Gly resin

Boc-Gly resin (Gly content: 2.2 millimoles) is placed in the reaction vessel of an automatic peptide synthesizer Shi-madzu APS-800, after which the following procedure is carried out in succession:

(1) Wash with dichloromethane (2 minutes, three times)

(2) 50% trifluoroacetic acid in dichloromethane (10 minutes, twice)

(3) Wash with dichloromethane (2 minutes, three times)

(4) Washing with ethanol (2 minutes, three times)

(5) Wash with chloroform (2 minutes, three times)

(6) chloroform containing 10% triethylamine (2 minutes and 10 minutes, once each)

(7) Wash with chloroform (2 minutes, three times)

(8) Condensation reaction with 6.6 millimoles of the symmetrical anhydride of BOC-amino acid (60 minutes) or 6.6 millimoles of BOC-amino acid p-nitrophenyl ester (12 hours for BOC-Asn, Boc-Gln or Boc-Tyr).

(9) Wash with dichloromethane (2 minutes, three times)

(10) acetylation of the unreacted amino group (dichloromethane containing 4.5% acetic anhydride)

(11) Wash with dichloromethane (2 minutes, three times)

After all reaction cycles have been carried out,

the product is washed in the order given with acetic acid, dimethylformamide and methanol and dried.

Yield: 13.15 g.

2) Preparation of the compound of the formula

Boc-Cys (MOB) -Tyr-Ile-Gln-Asn-Cys ~ (MOB) -Pro-Leu-Gly-NH2

12.6 g of the resin obtained under 1) are suspended in 70 ml of methanol containing 15.5% ammonia, whereupon the suspension is stirred for 46 hours at room temperature.

The resin is filtered off and washed twice with 20 ml of dimethylformamide. The filtrate and the washing liquids are combined and evaporated to dryness under reduced pressure. 50 ml of ether are added to the residue, and the resulting powder is isolated by filtration and dried. Yield 2.86 g. This product is purified by repeated reprecipitation from hot methanol, aqueous ethanol and a mixture of dimethylformamide and ethanol.

Yield 1.0 g.

io This powder is developed on a column (2.8 X 4.0 cm) with silica gel (solvent system: chloroform / methanol / water / pyridine / acetic acid = 1085: 150: 25: 63: 84).

The fractions rich in the desired compound are pooled and evaporated to dryness.

15

Yield: 768 mg; [a] o -39.1 ° (c = 0.47 in dimethylformamide);

Elemental Analysis for C66H98Q16N12S2 • CH3COOH-H20: Calculated: C55.52; H6.92; N 11.78; S4.49. 20 Found: C 55.24; H 7.00; N 11.92; S 4.43.

3) Production of oxytocin

125 mg of the powder prepared under 2) are dissolved together with 0.22 ml of anisole in 1 ml of trifluoroacetic acid, whereupon to 25 mg of Hg (CH3COO) 2 are added. The mixture is allowed to stand at room temperature for 30 minutes. After adding 30 ml of ether, the resulting precipitate is isolated by filtration (173 mg). This precipitate is dissolved in 3 ml of 50% aqueous acetic acid; after the addition of 30 0.31 ml of 2-mercaptoethanol, the resulting precipitate is filtered off. The filtrate is evaporated to dryness under reduced pressure. The residue is dissolved in 1 ml of 50% aqueous acetic acid and the solution is placed on a column (2.2 X 92.5 cm) with Sephadex G-15, whereupon it is eluted with 35 50% acetic acid. Fractions 149 to 188 ml are combined and freeze-dried (85 mg).

The freeze-dried product is dissolved in 150 ml of 0.1% aqueous acetic acid; after adjusting the solution to pH = 7.3 with 1-normal aqueous ammonia, air 40 is bubbled into the solution. After 4.5 hours the solution is brought to pH = 3.5 and freeze-dried (76 mg). The freeze-dried product is developed on a column similar to that mentioned above. Fractions from 154 to 188 ml are collected and freeze-dried to give 50 mg of oxytocin 45.

[a] 22-24.6 ° (c = 0.47.1 normal aqueous solution of acetic acid; amino acid analysis: Asp 1.03, Glu 1.13, Pro 1.06, Gly 1.00, Cys 1.97 , Ile 1.03, Leu 1.03, Tyr 0.90; thin layer chromatography: Rf (n-butanol / acetic acid / water = 3; 50 l: l) = 0.44; Rf (n-butanol / ethyl acetate / acetic acid / Was-ser = 1: 1: 1: 1) = 0.66 (in each case a single stain).

Example 5 Preparation of Somatostatin 55 1) Synthesis of the compound of the formula:

MOB

I.

H-Cys-O'Bu • L-tartrate

60

A suspension of 4.82 g (0.02 mol) of the compound of the formula:

MOB

65,

H-Cys-OH

in 150 ml of dioxane is added dropwise with stirring at room temperature

7

628 330

added 4.8 ml of concentrated sulfuric acid. 75 ml of isobutylene are bubbled into the mixture with ice cooling. The mixture is tightly sealed and allowed to stand at room temperature for 12 hours. The mixture is poured onto 300 ml of a 1 normal sodium hydroxide solution and extracted three times with ether. The ether layer is dried over anhydrous sodium sulfate and the ether is distilled off under reduced pressure. The oily residue is dissolved in 50 ml of ether, whereupon 3 ml of 6.69 normal hydrochloric acid in dioxane are added. The resulting crystals are isolated by filtration and recrystallized from ethyl acetate.

Yield: 2.4 g.

1.99 g of the crystals are suspended in ether and 10 ml of a 1 normal sodium hydroxide solution are added. The mixture is shaken so well that everything dissolves and extracted twice with ether. The ether solutions are combined, dried and distilled under reduced pressure. The oily residue is dissolved in ethanol. 900 mg L-tartaric acid are dissolved in the solution, after which it is left to stand for a while. The resulting crystals are isolated by filtration and recrystallized twice from ethanol.

Yield: 2.3 g; Melting point: 100.0 to 102.0 0C; [ci] d7 - 3.2 "(c = 0.53, dimethylformamide);

Elemental analysis for C15H2303NS.C4H606.1 / 2H20:

Calculated: C 49.99; H 6.62; N3.07; S 7.02. Found: C 49.85; H 6.74; N2.98; S 6.68.

2) Synthesis of the compound of the formula Z-Thr-Ser-OMe

6.22 g (0.04 mol) of the compound of the formula H-Ser-OMe-HCl are dissolved in 50 ml of dimethylformamide, whereupon triethylamine is added with ice-cooling. The resulting salt is separated by filtration, whereupon 16.6 g (0.04 mol) of the compound of the formula Z-Thr-ONB are added to the filtrate. The mixture is stirred at room temperature for 12 hours, whereupon the insoluble material is removed by filtration and the filtrate is concentrated in vacuo. The oily residue is dissolved in ethyl acetate, saturated with sodium chloride and washed with 1 normal hydrochloric acid and 5% sodium hydrogen carbonate. After drying, the ethyl acetate is distilled off, whereupon the resulting crystals are isolated.

Yield: 10.6 g (74.5%); Melting point: 133.0 to 135.0 ° C; [° 0d + 10.1 ° (c = 0.86, dimethylformamide);

Elemental analysis for C16H2207N2:

Calculated: C 54.23; H 6.26; N7.91.

Found: C 54.44; H 6.33; N7.84.

3) Synthesis of the compound of the formula Z-Phe-Thr-Ser-OMe

9.91 g (0.028 mol) of the compound of the formula Z-Thr-Ser-OMe are dissolved in 100 ml of methanol, after which 6.3 ml of 6.69 normal hydrogen chloride in dioxane are added. The mixture is subjected to catalytic reduction in the presence of palladium black in a conventional manner. The catalyst is filtered off and the filtrate is concentrated. The residue is dissolved in 50 ml of dimethylformamide and the solution is neutralized with 4.31 ml of triethylamine, after which it is filtered. The filtrate is mixed with the compound of the formula Z-Phe-ONB, which consists of 8.38 g (0.028 mol) of the compound of the formula Z-Phe-OH, 6.0 g (0.034 mol) of HONB and 5.78 g ( 0.028 mol) of dicyclohexylcarbodiimide is prepared, whereupon the mixture 12

Stir for hours at room temperature. The solvent is distilled off in vacuo and the residue is dissolved in ethyl acetate, followed by washing and drying in a conventional manner. Crystals separate out when the solvent evaporates. After cooling, the crystals are isolated by filtration.

Yield: 11.0 g (80.0%). Melting point: 180.0 to 182.0 ° C; [gc] d3-3.8 ° (c = 0.67, dimethylformamide);

Elemental analysis for C25H3108N3 • 1 / 2H20 Calculated: C 58.81; H 6.12; N8.23;

Found: C 58.80; H 6.25; N8.26.

4) Synthesis of the compound of the formula

Z (N02) -Thr-0H • DCHA

The connection is carried out according to the conventional method [F. H. Carpenter and D. T. Gish, J. Amer. Chem. Soc. 74, 3818 (1952)] with sodium bicarbonate as the base.

Yield: 50.0%; Melting point: 186.0 to 188.0 ° C; [o.] q + 14.9 ° (c = 0.77, dimethylformamide);

Elemental analysis for CpH14N207 • C12H23N Calculated: C60, ll; ~ H7.78; N8.76;

Found: C 60.14; H 7.83; N8.80.

5) Synthesis of the compound of the formula

Z (N02) -Thr-Phe-Thr-Ser-0Me

According to a conventional method, 11.0 g (0.022 mol) of the compound of the formula Z-Phe-Thr-Ser-OMe in methanol are catalytically reduced to H-Phe-Thr-Ser-OMe. A solution of the compound of the formula (Z (NO 2) -Thr-0NB in tetrahydrofuran, which consists of 10.6 g (0.022 mol) of the compound of the formula Z (NO 2) -Thr-OH • DCHA, 3 , 94 g (0.022 mol) of HONB and 4.54 g (0.022 mol) of dicyclohexylcarbodiimide, the mixture is stirred at room temperature for 12 hours, the solvent is distilled off, water is added and the resulting crystals are isolated by filtration and isolated Recrystallized methanol.

Yield: 12.8 g (90.1%); Melting point: 194.0 to 196.0 ° C; Md - 057 ° (c = 0.72, dimethylformamide);

Elemental Analysis for C29H37012N5 Calculated: C 53.78; H 5.76; N 10.81;

Found: C 53.80; H 5.85; N 10.75.

6) Synthesis of the compound of the formula

Z (N02) -Thr-Phe-Thr-Ser-NHNH2

12.3 g (0.019 mol) of the compound of the formula Z (NO 2) -Thr-Phe-Thr-Ser-OMe are dissolved in 100 ml of dimethylformamide, then 100 ml of methanol and then 9.5 ml (0.19 mol) Hydrazine hydrate added. The mixture is left to stand at room temperature for 48 hours and cooled with ice. The resulting crystals are isolated by filtration and recrystallized from methanol.

Yield: 11.4 g (92.7%); Melting point: 229.0 to 232.0 ° C (decomposition); [a] o + 3.2 ° (c = 0.44, dimethylformamide); Elemental analysis for C28H37OnN7 • H20 Calculated: C 50.52; H 5.91; N 14.81;

Found: C 50.89; H 5.92; N 14.90.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

628 330

8th

7) Synthesis of the compound of the formula:

MOB

I.

Z (N02) -Thr-Phe-Thr-Ser-Cys-0tBu

3.24 g (0.005 mol) of the compound of the formula Z (NO 2) -Thr-Phe-Thr-Ser-NHNH2 are dissolved in 50 ml of dimethylformamide. While the solution is cooled to -20 ° C, 3.1 ml of 6.51 normal hydrogen chloride in dioxane are added and 0.81 ml (0.06 g mol) of isoamyl nitrite is added dropwise. After 10 minutes the mixture is further cooled to -50 ° C and neutralized with triethylamine.

In the meantime, 2.24 g (0.005 mol) of the compound of the formula:

MOB

l

H-C ys-O'B u-L-tartrate dissolved in dimethylformamide and the solution neutralized with ice-cooling with 1.4 ml of triethylamine. This solution is combined with the azide solution prepared above and the combined solutions are stirred at 4 ° C for 48 hours. The insoluble material is removed by filtration and the filtrate is concentrated. Water is added to the residue and the resulting powder is isolated by filtration. The powder is recrystallized twice from methanol.

Yield: 3.55 g (77.7%); Melting point: 193.0 to 195.0 ° C (decomposition); [a] gs -18.0 ° (c = 0.56, dimethylformamide); Elemental Analysis for C43H56Oi4N6S Calculated: C56.67; H6.18; N9.21; S 3.51; Found: C 56.31; H 6.08; N9.34; S 3.47.

8) Synthesis of the compound of the formula

Z (NOz) -Trp-OH

The connection is made by a conventional method.

Yield: 88.6%; Melting point: 108.0 to 115.0 ^ (decomposition); [a] p -43.5 ° (c = 0.65, dimethylformamide); Elemental analysis for C19H1704N3 • 2H20 Calculated: C 58.91; H 5.46; N 10.85;

Found: C 59.63; H 4.63; N 10.74.

9) Synthesis of the compound of the formula:

BOC

I.

Z (N 02) -Trp-L ys-OH • DCHA

7.39 g (0.03 mol) of the compound of the formula:

BOC

I.

H-Lys-OH

are dissolved in 100 ml of dimethylformamide, whereupon 4.2 ml (0.03 mol) of triethylamine are added. The solution is mixed with the compound of the formula Z (N02) -Trp-ONB, which consists of 10.5 g (0.03 mol) of the compound of the formula Z (N02) -Trp-0H, 5.4 g (0, 03 mol) of HONB and 6.19 g (0.03 mol) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours, after which the solvent is distilled off. After adding 30 ml of 1N hydrochloric acid, the mixture is dissolved in ethyl acetate. The ethyl acetate layer is washed three times with water, dried and distilled to evaporate the solvent. The oily residue is dissolved in 100 ml of acetonitrile and mixed with 6.0 ml (0.03 mol) of dicyclo-

5 hexylamine added, whereupon the mixture is left in a refrigerator. The resulting gelatinous crystals are isolated by filtration and recrystallized from acetonitrile.

io yield: 12.5 g (52.6%); Melting point: 95.0 to 100.0 ° C; [oi] q - 20.9 ° (c = 0.56, dimethylformamide); Elemental analysis for C30H37O9N5 • C12H23N Calculated: C 63.61; H 7.63; N 10.60;

Found: C 63.77; H 7.67; N 11.08.

10) Synthesis of the compound of the formula:

BOC MOB

20 I I

Z (NQ2) -Trp-Lys-Thr-Phe-Thr-Ser-Cys-OtBu

3.29 g (0.0036 mol) of the compound of the formula:

25 MOB

I.

Z (NO2) -Thr-Phe-Thr-Ser-Cys-O'Bu are dissolved in 150 ml of a mixture of acetic acid and water 30 (volume ratio 8: 2), followed by the solution at 50 ° C for 8 hours of catalytic reduction in the presence of palladium black. The catalyst is filtered off and the filtrate is freeze-dried. The freeze-dried product is dissolved in methanol and ether is added. The resulting precipitate is isolated by filtration and reprecipitated from a mixture of methanol and ether. Yield: 2.75 g

2.62 g (0.0033 mol) of the powder obtained above are dissolved in a small amount of dimethylformamide. A solution 40 of 627 mg of p-toluenesulfonic acid in dimethylformamide is added to the solution with cooling, whereupon the mixture is allowed to stand. Most of the dimethylformamide is distilled off and ether is added to the residue. The resulting powder is isolated by filtration and dissolved in 20 ml of Dime-45 thylformamide, whereupon the compound of the formula:

BOC I

Z (NO2) -Trp-Lys-OH

50

who adds from 2.88 g (0.0036 mol) of the compound of formula:

BOC

I.

55 Z (NOz) -Trp-Lys-OH • DCHA

is prepared, and further 1.30 g (0.0072 mol) of HONB, 0.42 ml (0.0033 mol) of N-ethylmorpholine and 819 mg (0.004 mol) of di-cyclohexylcarbodiimide are added. The mixture is stirred at -10 ° C for 60 hours and stirred at room temperature for a further 48 hours. The resulting dicyclohexyl urea is filtered off and the filtrate is concentrated. Water is added to the residue.

The resulting precipitate is isolated by filtration 65 and recrystallized twice from methanol.

Yield: 3.5 g (80.6%); Melting point: 183.0 to 185.0 ° C (decomposition);

9

628 330

[a] 0-26.6 ° (c = 0.67, dimethylformamide); Elemental analysis for N10S

Calculated: C 58.52; H 6.45; N 10.66; S 2.44;

Found: C 58.36; H 6.47; N 10.49; S 2.54.

11) Synthesis of the compound of the formula Z-Phe-Phe-O'Bu According to a conventional method, 17.8 g (0.05 mol) of the compound of the formula Z-Phe-O'Bu in methanol become catalytically the compound of the formula H-Phe-O'Bu reduced. This compound is mixed with a solution of the compound of the formula Z-Phe-ONB, which consists of 15.0 g (0.05 mol) of the compound of the formula Z-Phe-OH, 9.0 g (0.05 mol) of HONB and 10.3 g (0.05 mol) of dicyclohexylcarbodiimide is prepared, and the mixture is stirred at room temperature for 12 hours. The dioxane is distilled off and the residue is dissolved in ethyl acetate. The solution is washed, dried and distilled in a conventional manner to evaporate the ethyl acetate. The resulting crystals are isolated by filtration and recrystallized from ethyl acetate.

Yield: 15.4 g (60.0%); Melting point: 107.0 to 109.0 ° C; [a] o -13.5 ° (c = 0.53, dimethylformamide);

Elemental Analysis for C30H34O5N2 Calculated: C 71.96; H 6.82; N5.57;

Found: C 71.61; H 6.64; N5.50.

12) Synthesis of the compound of the formula BOC-Asn-Phe-Phe-O'Bu

Conventionally, 13.1 g (0.026 mol) of the compound of the formula Z-Phe-Phe-O'Bu in methanol are catalytically reduced to the compound of the formula H-Phe-Phe-O'Bu. This compound is dissolved together with 6.04 g (0.026 mol) of the compound of the formula BOC-Asn-OH and 4.65 g (0.026 mol) of HONB in 50 ml of dimethylformamide. 5.36 g (0.026 mol) of dicyclohexylcarboiimide are added with ice cooling, and the mixture is stirred for 3 hours at 0 ° C. and for a further 12 hours at room temperature. The resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo, after which water is added. The resulting crystals are isolated by filtration and recrystallized twice from methanol.

Yield: 11.0 g (72.8%); Melting point: 163.0 to 166.0 ° C; [a] j) 7-28.9 ° (c = 0.85, dimethylformamide); Elemental Analysis for C31H4207N4 Calculated: C63.90; H 7.27; N9.62;

Found: C 64.12; H 7.20; N9.90.

BOC I

Z-Lys-ONB

5, which consists of 8.2 g (0.0216 mol) of the compound of the formula:

BOC

I.

io Z-Lys-OH

3.8 g (0.0216 mol) of HONB and 4.4 g (0.0216 mol) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours, after which 10 ml of acetic acid are added to -15. The solvent is distilled off in vacuo and water is added to the residue. The resulting precipitate is isolated by filtration and purified by reprecipitation twice from aqueous methanol and then from a mixture of dimethylformamide and ethyl acetate.

20th

Yield 11.1 g (78.1%); Melting point: 194.0 to 196.0 ° C (decomposition); [a] "- 26.9 ° (c = 0.59, dimethylformamide); elemental analysis for C41H52O10N6 calculated: C 62.42; H 6.64; N 10.65;

25 Found: C 62.27; H 6.66; N10.72.

14) Synthesis of the compound of the formula:

30th

MOB

I.

Z (N02) -Cys-0H

The connection is made by a conventional method.

35

Yield: 80.0%; Melting point: 163.0 to 165.0 ° C; Md - 51.2 ° (c = 0.61, dimethylformamide); Elemental Analysis for C19H20O7N2S Calculated: C 54.28; H 4.79; N6.66; S 7.62; 40 Found: C54.18; H4.74; N6.57; S7.56.

15) Synthesis of the compound of the formula:

MOBBOC

45

50

13) Synthesis of the compound of the formula:

BOC

!

Z-Lys-Asn-Phe-OH

10.5 g (0.018 mol) of the compound of the formula BOC-Asn-Phe-Phe-O'Bu are dissolved in 100 ml of trifluoroacetic acid, and the mixture is then left to stand at room temperature for 1.5 hours. The trifluoroacetic acid is distilled off under reduced pressure and ether is added to the residue. The resulting precipitate is isolated by filtration and dried and dissolved in 100 ml of dimethylformamide. The solution is mixed with 5.04 ml (0.036 mol) of triethylamine and a solution of the compound of the formula:

55

65

Z (N02) - Cys - Lys - Asn - Phe - Phe - OH

According to a conventional method, 10.3 g (0.013 mol) of the compound of the formula:

BOC

I.

Z-Lys-Asn-Phe-Phe-OH

catalytically reduced in a mixture of acetic acid and water (volume ratio 8: 2). The resulting compound of the formula:

BOC

I.

H-Lys-Asn-Phe-Phe-OH

is purified by reprecipitation from ethanol twice. Yield: 7.0 g.

6.55 g (0.01 mol) of this compound are suspended in 100 ml of dimethylformamide, then 1.40 ml (0.01 mol) of triethylamine and further a solution of the compound of the formula:

628 330

10 '

MOB

I.

Z (N02) -Cys-0NB

in dimethylformamide, which consists of 5.05 g (0.012 mol) of the compound of the formula:

MOB

i

Z (NO2) -C ys-OH

2.15 g (0.012 mol) of HONB and 2.48 g (0.012 mol) of dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours. After adding 10 ml of acetic acid, the dimethylformamide is distilled off in vacuo and water is added to the residue. The resulting precipitate is isolated by filtration and recrystallized twice from a mixture of methanol and water (volume ratio 8: 2).

Yield: 9.6 g (92.3%); Melting point: 209.0 to 210.0 ° C (decomposition); [a7-24.6 ° (c = 0.74, dimethylformamide); Elemental Analysis for C52H64014N8S Calculated: C 59.08; H 6.10; N 10.60; S 3.03; Found: C59.04; H6.02; N 10.60; S 3.19.

16) Synthesis of the compound of the formula BOC-Ala-Gly-OBzl

8.10 g (0.024 mol) of the compound of the formula H-Gly-OBzl • p-Ts-OH are dissolved in 50 ml of dimethylformamide, and 3.36 ml (0.024 mol) of triethylamine are added while cooling with ice. The mixture obtained is mixed with the compound of the formula BOC-Ala-ONB, which consists of 4.35 g (0.023 mol) of the compound of the formula BOC-Ala-OH, 4.12 g (0.023 mol) of HONB and 4.74 g (0.023 mol) dicyclohexylcarbodiimide. The mixture is stirred at room temperature for 12 hours and the solvent is distilled off in vacuo. The residue is dissolved in ethyl acetate, washed in a conventional manner and dried. The ethyl acetate is distilled off and petroleum ether is added to the residue in order to crystallize it. The crystals are isolated by filtration and recrystallized from a mixture of ethyl acetate and petroleum ether.

Yield: 5.5 g (71.2%); Melting point: 86.0 to 87.0 ° C; [aj7-10.8 ° (c = 0.56, dimethylformamide);

Elemental Analysis for C17H2405N2 Calculated: C 60.70; H 7.19; N8.33;

Found: C 60.82; H 7.19; N8.28.

17) Synthesis of the compound of formula

BOC-Ala-Gly-OH

5 According to a conventional method, this compound is prepared by catalytic reduction of 5.29 g (0.015 mol) of the compound of the formula BOC-Ala-Gly-OBzl in methanol.

io yield: 3.3 g (85.3%); Melting point: 111.0 to 113.0 ° C (decomposition); [a] ^ 7-6.8 ° (c = 0.56, dimethylformamide); Elemental analysis for C10H18O5N2 • 1 / 2H20 Calculated: C47.05; H 7.50; N 10.98;

Found: C46.66; H 7.65; N 11.03.

15

18) Synthesis of the compound of the formula:

MOBBOC I I

20 BOC - Ala - Gly - Cys - Lys - Asn - Phe - Phe - OH 5.2 g (0.005 mol) of the compound of the formula:

MOBBOC

25 | |

Z (N02) - Cys-Lys - Asn - Phe - Phe - OH

are reduced catalytically in a mixture of acetic acid and water (volume ratio 8: 2) over palladium black for 8 hours at 30 50 ° C. The catalyst is filtered off and the filtrate is concentrated, whereupon the residue is triturated with ether. The resulting powder is isolated by filtration and reprecipitated from ethanol. Yield: 4.05 g.

The powder is suspended in 50 ml of dimethylformamide and mixed with 0.7 ml of triethylamine and then with the compound of the formula BOC-Ala-Gly-ONB, which consists of 1.48 g (0.006 mol) of the compound of the formula BOC-Ala- Gly-OH, 1.08 g (0.006 mol) HONB and 1.24 g (0.006 mol) dicyclohexylcarbodiimide. The mixture is stirred vigorously for 12 hours. After acidification with 10 ml of acetic acid, the dimethylformamide is distilled off in vacuo. Water is added to the residue and the resulting powder is isolated by filtration and reprecipitated twice from a mixture of methanol and water (volume ratio 8: 2).

45

Yield: 3.5 g (60.4%); Melting point: 201.0 to 203.0 ° C (decomposition); [ajo - 28.2 ° (c = 0.69, dimethylformamide); Elemental analysis for Cs ^ - ^ O ^ NgS Calculated: C58.63; H6.83; Nil, 40; S2.90; 50 Found: C58.86; H6.83; N 11.53; S2.79.

19) Synthesis of the compound of the formula:

MOB BOC BOC MOB

II I • I

BOC - Ala - Gly - Cys - Lys - Asn - Phe - Phe - Trp - Lys - Thr - Phe-Thr - Ser-Cys - O'Bu

3.29 g (0.0025 mol) of the compound of the formula:

BOC MOB

I I

Z (NO2) -Trp-Lys-Thr-Phe-Thr-Ser-Cys-O'Bu are suspended in 150 ml of a mixture of acetic acid and water (volume ratio 8: 2), whereupon the compound at 50 ° C. 5 Is reduced for hours over palladium black. The catalyst is removed by filtration and the filtrate is concentrated under reduced pressure. A small amount of ethanol is added to the residue, and ether is also added to triturate the residue. This process is repeated.

Yield: 2.90 g.

2.69 g (0.0023 mol) of the compound prepared above

11

628 330

are dissolved in a small amount of dimethylformamide and a solution of 437 mg (0.0023 mol) of p-toluenesulfonic acid in dimethylformamide is added dropwise while cooling with ice. After allowing the mixture to stand for a while, the solvent is distilled off in vacuo and ether is added to triturate the residue. The resulting powder is dissolved in 20 ml of dimethylformamide, followed by 823 mg (0.0046 mol) of HONB, 0.29 ml (0.0023 mol) of N-ethylmorpholine and 2.54 g (0.0023 mol) of the compound of Formula:

MOBBOC

I (

BOC - Ala - Gly - Cys - Lys - Asn - Phe - Phe - OH

be added. At -10 ° C, 949 mg (0.0046 mol) of di-cyclohexylcarbodiimide in dimethylformamide are added, after which the mixture is stirred for 3 hours at the same temperature and for a further 72 hours at room temperature. The

The resulting dicyclohexylurea is removed by filtration and the filtrate is concentrated in vacuo. Water is added to the residue and the resulting precipitate is isolated by filtration and reprecipitated twice from methanol.

io yield: 3.8 g (75.1%); Melting point: 224.0 to 226.0 ° C (decomposition); [aj -18.3 ° (c = 0.70, dimethylformamide); Elemental Analysis for QogH ^ OzeN ^ Sz Calculated: C59.62; H7.06; Nil, 47; S2.92; Found: C 59.55; H 6.75; N 11.32; S 2.95.

15

20) Synthesis of the compound of the formula:

I I

H - Ala - Gly - Cys - Lys - Asn - Phe - Phe - Trp - Lys — Thr - Phe - Thr - Ser - Cys - OH (Somatostatin)

220 mg (0.1 mmol) of the compound of the formula:

MOBBOC BOC MOB

II I I

BOC - Ala - Gly - Cys - Lys - Asn - Phe - Phe - Trp - Lys - Thr - Phe-Thr - Ser - Cys - O'Bu are shaken in 20 ml of a mixture of acetic acid and water solution at room temperature for 30 minutes . The

(Volume ratio 8: 2) solved. The solution is mixed with 171 mg of 30 trifluoroacetic acid at 30 ° C under reduced pressure

(0.4 mmol) of Hg (CF3COO) 2 was added and the mixture was distilled off over the course of 10 minutes. Ether is added

Stir room temperature for 12 hours. Sulfur and the resulting precipitate are isolated by filtration and hydrogen gas are bubbled into the reaction mixture, and the solution is dissolved in a small amount of water. The solution is separated by resulting black precipitate by filtration with Ce- a column (2.0 X 7.0 cm) with Amberlite IRA-410 (acetate form)

lite. The filtrate is concentrated under reduced pressure and passed to carry out the ion exchange. The outflowing the residue is triturated with ether, which is a small amount of liquid (60 ml) through a column (5.5 X 35.0 cm)

contains ß-mercaptoethanol. The resulting powder is passed to Sephadex LH-20, which is mixed with 0.1 normal acetic acid solution.

isolated by filtration and dried. solution is eluted. The main fractions from 560 to 640 ml are

Yield: 193 mg. the caught and freeze-dried.

The compound is dissolved in 500 ml of dimethylformamide, 40

whereupon 0.28 ml of 10% triethylamine-containing dimethylfor yield: 20.4 mg; [<x] d - 31.2 ° (c = 0.13, 1% vinegar-mamide and further 30 mg of 1,2-diiodoethane are added. The acid); Thin layer chromatography: Rf = 0.60 (n-butanol /

Reaction mixture is pyridine / acetic acid / water in a volume ratio of 30: 20: 6: 24 for 30 minutes at room temperature.

stirred and the solvent distilled off in vacuo. The Avicel), Rf = 0.40 (n-butanol / ethyl acetate / acetic acid / water

The residue is triturated with ether and the resulting pulp 45 in a volume ratio of 1: 1: 1: 1, silica gel);

isolated by filtration and dried. Amino acid analysis (6 normal hydrochloric acid, 110 ° C, 24 hours):

Yield: 158 mg. Lys 1.97 (2); Asp 1.01 (1); Thr 1.95 (2); Ser 0.92 (1); Gly

The compound is dissolved in 3 ml of a mixture of trifluo- 1.00 (1); Ala 1.02 (1); Half Cys 1.58 (2); Phe 3.01 (3); by-

vinegar and water (volume ratio 9: 1) dissolved and the average recovery 82%.

C.

Claims (8)

628 330 2nd PATENT CLAIMS can be removed in good yield by using the sub- 1. Procedure for removing one or more protective layers treated under very mild conditions, i.e. with a group (s) from an amino acid or a peptide with a mercury salt of a carboxylic acid of the formula: or more thiol group (s) which are formed by p-methoxybenzyl, 1 -Adamantyl or tert-butyl is or are protected, characterized by 5 CX3COOH (I), that the protected amino acid or the protected peptide with the mercurium salt of a carboxylic acid in which X is hydrogen, chlorine or fluorine. Formula CX3COOH, where X is hydrogen, chlorine or fluorine. The present invention thus relates to a meaning. extend to remove one or more protective group (s) 2. The method according to claim 1, characterized in 10 an amino acid or a peptide with one or more that one uses the mercuric salt of acetic acid. Thiol group (s), which can be substituted by p-methoxybenzyl, 1-adamantyl or 3. The method according to claim 1, characterized in that tert-butyl is or are protected, which method by using the mercuric salt of trifluoroacetic acid. is characterized in that the protected amino acid or 4. The method according to claim 1, characterized in that the protected peptide with a mercuric salt of a carboxylic acid that the mercuric salt of trichloroacetic acid is used. 15 of formula I treated. 5. The method according to any one of claims 1 to 4, characterized in that the present method is applicable to any characterized in that protected amino acids with one or more thiol group (s), the tes cysteine is used as the protected amino acid. is protected by one or more of these protective groups 6. The method according to any one of claims 1 to 4, thereby or are; Examples of such amino acids are cysteine and Ho-labeled using a protected peptide, the 20 mocysteine. The present method is also applicable to one or more cysteine residues. any peptides with one or more thiol group (s) which 7. The method according to any one of claims 2, 5 and 6, characterized by one or more of these protective groups, characterized in that the removal of the protective groups in these peptides can be those which one or more presence of the mercuric salt of acetic acid in trifluoroacetic acid Residue (s) of homocysteine or any other that carries an acid. Amino acid containing 25 or more thiol group (s) as 8. The method according to any one of claims 3, 5 and 6, characterized by building block, the thiol group (s) being protected by one or by protecting the removal of the protective groups into several of these protective groups; a typical presence of the mercuric salt of trifluoroacetic acid in the vinegar of such a peptide is glutathione. The present acid exports. The method can also be used for the production of ami 30 nucleic acids or peptides with one or more disulfide linkage (s), e.g. Cystine, oxytocin, vasopressin, somatostatin, insulin, calcitonin, trypsin inhibitor, ribonuclease or lysozyme.